title: Preparation of developing Xenopus muscle for sarcomeric protein localization by high-resolution imaging.
abstract: Mutations in several sarcomeric proteins have been linked to various human myopathies. Therefore, having an in vivo developmental model available that develops quickly and efficiently is key for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; this is the pivotal foundation for deciphering disease mechanisms as well as the development of myopathy-related therapeutics. Although striated muscle cell culture studies have been extremely informative in providing clues to both the distribution and functions of sarcomeric proteins, myocytes in vivo develop in an irreproducible 3D environment. Xenopus laevis (frog) embryos are cost effective, compliant to protein level manipulations and develop relatively quickly (⩽ a week) in a petri dish, thus providing a powerful system for de novo myofibrillogenesis studies. Although fluorophore-conjugated phalloidin labeling is the gold standard approach for investigating actin-thin filament architecture, it is well documented that phalloidin-labeling can be challenging and inconsistent within Xenopus embryos. Therefore we highlight several techniques that can be utilized to preserve both antibody and fluorophore-conjugated phalloidin labeling within Xenopus embryos for high-resolution fluorescence microscopy.
authors.affiliation: Department of Cellular and Molecular Medicine and The Sarver Molecular Cardiovascular Research Program, The University of Arizona, Tucson, AZ 85724, USA.
authors.forename: Chinedu U
authors.initials: CU
authors.lastname: Nworu
authors.affiliation: Department of Cellular and Molecular Medicine and The Sarver Molecular Cardiovascular Research Program, The University of Arizona, Tucson, AZ 85724, USA.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Krieg
authors.affiliation: Department of Cellular and Molecular Medicine and The Sarver Molecular Cardiovascular Research Program, The University of Arizona, Tucson, AZ 85724, USA. Electronic address: gregorio@email.arizona.edu.
authors.forename: Carol C
authors.initials: CC
authors.lastname: Gregorio
mesh_terms: D000818:Animals; D016616:Cryoultramicrotomy; D008856:Microscopy, Fluorescence / Q000379:methods*; D024510:Muscle Development; D010590:Phalloidine / Q000032:analysis; D012518:Sarcomeres / Q000378:metabolism*; D016707:Tissue Fixation; D029867:Xenopus Proteins / Q000032:analysis*; D014982:Xenopus laevis
chemical_list: D029867:Xenopus Proteins; D010590:Phalloidine
keywords: Immunofluorescence microscopy; Myofibrillogenesis; Phalloidin; Sarcomere; Xenopus laevis